Bioluminescent imaging (BLI) with firefly luciferase has gained widespread acceptance as a powerful, inexpensive, and non-invasive method to monitor gene expression, enzymatic activity, protein-protein interactions and protein degradation in the context of the whole organism. Relative to other imaging modalities such as PET and MRI, bioluminescence imaging has the advantages of low cost, speed, sensitivity, high throughput and ease of use by non-specialists. These advantages make BLI the method of choice for rapidly assessing tumor progression and response to potential therapeutics. The major limitation of BLI is the poor penetration of visible light through tissue. Illuminated tissue is most transparent to near-IR light (650-900 nm), where autofluorescence, scattering, and the absorption of visible light by hemoglobin is minimal. Despite considerable effort spent isolating and mutagenizing luminescent proteins, there is no luciferase that maximally emits light >650 nm. We propose two synergistic approaches to shift the light output of firefly luciferase to the near-IR: 1) Resonance energy transfer to a targetable near-IR fluorophore, and 2) Synthesis of novel luciferin substrates that maximally emit light at longer wavelengths. These aims are relevant to in vivo cancer imaging because they will improve the speed, detection limit, and depth penetration of bioluminescence imaging. Introduction of cancer cells that express light-emitting proteins into mice has allowed researchers to monitor cancer progression and response to drug treatment in living animals. However, the emitted light is strongly absorbed by blood, and thus does not travel far through the mouse. We are changing the wavelength of the emitted light to avoid absorption by blood and allow deeper penetration of the light through the mouse, improving our ability to detect small tumors or tumors in deep organs.